Article pubs.acs.org/ac
High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions Yuchen Pan,† Todd A. Duncombe,† Colleen A. Kellenberger,‡ Ming C. Hammond,‡,§ and Amy E. Herr*,† †
Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, Berkeley, California 94720, United States ‡ Department of Chemistry, University of California Berkeley, Berkeley, California 94720, United States § Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 94720, United States S Supporting Information *
ABSTRACT: We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mmscale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/ min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch−ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets.
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microfluidic FP and FRET have improved assay throughput but have not been applied to the study of riboswitches.8,9 Alternatively, electrophoretic mobility shift assays (EMSAs), introduced by Garner and Revzin,10 have played an important role in the study of numerous molecular binding interactions including riboswitches. Widely employed, EMSAs measure changes in the physical properties of a target analyte (i.e., size, charge, conformation) upon binding with a ligand. Bindinginduced changes can shift the electrophoretic mobility of the target analyte, a measurable quantity allowing quantitative characterization of the binding reaction.11,12 Yet, conventional EMSAs are conducted with slab polyacrylamide gel electrophoresis (PAGE), which is low-throughput and not suitable for
iboswitches are functional RNA molecules that play a key role in gene regulation of many important processes in diverse bacterial species.1 Located in the 5′ untranslated regions (UTRs) of mRNA, riboswitches directly bind to small molecule metabolites through their aptamer domain, triggering a conformational change that alters expression of the downstream gene.2 Owing to selective ligand binding, riboswitches have garnered interest as promising and largely unexplored antibiotic drug targets.3 To maximize the chance of discovering riboswitch-targeting antibacterial ligands, a rapid and robust screening platform is required to efficiently identify potential riboswitch-binding compounds from compound libraries.3,4 Gold-standard screening tools include fluorescence polarization (FP)5 and fluorescence resonance energy transfer (FRET).6 While powerful, FP and FRET are not readily implemented as high-throughput assays, as both are sample-intensive, are slow, and require substantial infrastructure.7 Recently, droplet © XXXX American Chemical Society
Received: July 21, 2014 Accepted: September 18, 2014
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screening assays.13,14 For example, binding of the Vc2 GEMM-I riboswitch to its ligand cyclic di-GMP (c-di-GMP), an important second messenger in bacteria,15 induces a significant structural compaction of the riboswitch aptamer.16 This compaction results in a higher electrophoretic mobility in the gel matrix than its free form. To electrophoretically resolve the bound and unbound RNAs via conventional slab-gel EMSAs requires up to 17 h.17 Capillary electrophoresis (CE) is an efficient separation modality and Stebbins et al.18 introduced CE for EMSAs to analyze binding between the trp repressor in Escherichia coli and the trp operator (DNA). The mobility shift was resolved in 6 min. To further advance the quantitative capacity and throughput of EMSAs, Karns and colleagues17 introduced microfluidic PAGE to study the binding characteristics of Sadenosylmethionine-I riboswitches. A key advantage of this microfluidic electrophoresis platform is the efficient heat dissipation due to a large surface area-to-volume ratio in microchannels. This favorable thermal transport characteristic enables the use of high electric field (e.g., 400 V/cm in-chip and ∼10 V/cm on slab PAGE), thus resolving binding pairs in 30 s (compared to 17 h on slab PAGE). However, regardless of the rapid analysis, the microchip format was limited to single and serial implementation of separations. As a result, the entire assay protocol (i.e., loading-separation-washing) required ∼3 min, limiting usefulness in high-throughput screening. A recent breakthrough in increasing analytical throughput takes advantage of multiplexed formats. In 2000, Ferrance et al.19 demonstrated an 8-plex multichannel microfluidic separation system for rapid clinical diagnostics. Later, Ross and colleagues20 demonstrated a 16-plex gradient elution moving boundary electrophoresis tool for monitoring enzyme activity and inhibition. Researchers have also introduced microfluidic systems compatible with powerful, conventional biological tools (e.g., a microplate format). For example, a 384-plex radial microfluidic capillary electrophoresis tool developed by Emrich et al.21 supported ultrahigh-throughput genetic analyses. In 2003, Gaunt et al.22 established a microplate gel array platform for SNP genotyping, in which a PAG molded with sample wells was seated on a silane-treated glass plate. With the molding strategy, 768 wells were fabricated in the PAG sheet, yielding the highest reported multiplexed electrophoresis tool to our knowledge. At the single-cell level, Gutzkow et al.23 developed a 96-plex minigel platform for high-throughput comet assays, using cell-suspended agarose droplets patterned on a piece of polyester film (Gel bond). Engelward and colleagues24,25 have developed the CometChip, a sheet of agarose gel patterned with microwells that house single cells. The CometChip has been applied to assess genotoxicity mediated by engineered nanoparticles. In 2014, we introduced a microfluidic device that enables completion of thousands of concurrent single-cell western blots.26 Building on our previous work which reported electrophoretic resolution of proteins in 96-plex fsPAG electrophoresis (fsPAGE) assay,27 we now address an unmet throughput need for riboswitch screening. We detail design considerations for successful introduction of multiplexed fsPAG-EMSAs. Screening for changes in riboswitch conformation presents new analytical challenges for an EMSA system. For context, our previous fsPAGE protein analyses measured large relative electrophoretic mobility shifts between adjacent peaks (i.e., 2× changes in the electrophoretic mobility) in a standard low conductivity buffer. Riboswitch mobility shifts are compara-
tively small, 10% relative mobility shifts (bound and unbound), and require high conductivity buffers, which limits the possible assay time before heating effects disrupt the assay. In this work, we quantitatively characterized the fsPAGE platform and optimized the system to improve sample injection and resolving power over short migration distances and assay times. Through optimization studies, we determined optimal conditions for performing high-throughput (80%. On the basis of these observations, we hypothesize that glycerol assists with maintaining fsPAG structural stability thus extending the effective electrophoresis time. To evaluate the influence of enhanced viscosity (reducing mobility and diffusion) on the separation performance, we performed fsPAGE across a range of glycerol conditions on H = 100 μm tall structures with fluorescent scalar TI*. For the conditions studied, the resultant TI* migration distance (L) and bandwidth (4σ) are reported in Figure 4C. As anticipated, increasing glycerol concentrations reduced both the TI* migration distance and bandwidth. To quantify the resolving
Figure 3. fsPAG geometry impacts current stability during electrophoresis. (A) Current stability over time during fsPAGE at E = 60 V/ cm. fsPAG of 70−100 μm preserves most conductivity. (B) Current drop measurement for a range of fsPAG structure heights at 3 min. The highest relative current (white data point) is observed at 100 μm.
400 μm) exhibited an initial ramp up in current followed by a steep decrease in current after ∼60 s of operation. The shorter fsPAG structures (H ≤ 200 μm) showed less dynamic behavior but did exhibit a slow reduction of current during operation. Especially evident in the large structures, we attribute the initial current rise to an increase in gel temperature owing to Joule heating which, in turn, affects evaporation and ion concentration, leading to increased conductivity. In accordance, we attribute the observed subsequent sharp decrease in current to physical collapse of the gel structures owing to dehydration. Dehydration shrinks the porous gel matrix, thus reducing the conducting cross-section of the fsPAG lane. During the period when fluid is still present, the increasing resistance would reduce the current. Given these observations, we sought to minimize the rate of current drop to improve EMSA performance stability over an anticipated EMSA duration (Figure 3B). Using estimates from enclosed microchannel EMSAs of the c-di-GMP riboswitch (mobility shift of 2.5 × 10−6 cm2/V s, work in preparation), a 3 min separation is anticipated to allow full resolution of the mobility shift between bound and unbound riboswitch (Figure 3B). At this 3 min time point, a fsPAG structure with H = 100 μm maintains ∼90% of the initial current value, whereas the H = 600 μm structure maintains just ∼40% of the initial current. Consequently, all E
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Figure 4. Glycerol addition improves fsPAGE operational stability. (A) Current dynamics of fsPAG structures with different glycerol concentrations. (B) Optical profilometry of gel height relative to initial structure height at set time points during fsPAGE. Initial height = 100 μm. (C) Measurements of migration distance, bandwidth, and their ratio at different glycerol concentrations during fsPAGE at 3 and 6 min. Protein: 500 nM OVA. (D) Fluorescence images of protein band migration during fsPAGE for 0% and 20% glycerol at 3 min during separation. E = 60 V/cm. fsPAG height = 100 μm.
power, we considered the ratio between migration distance and bandwidth (L/4σ) as an approximation for separation resolution in more complex samples. After 3 and 6 min of electrophoresis time, structures containing glycerol yielded notably higher resolving powers than structures containing no glycerol. To assess device reliability, we compared the band shape over three separate trials in structures with 0 and 20% glycerol concentrations. Figure 4D shows resultant images after 3 min of electrophoresis time, with dramatic band distortion and variation observed in the replicates with no glycerol present. Unsuitable repeatability and bad band shape control were likely caused by rapid evaporation, giving rise to nonuniform gel drying which leads to heterogeneity in analyte mobility across the gel. In contrast, structures containing higher glycerol concentrations (20%) yielded a consistent band with notably less transverse skew. Thus, run buffer containing 20% glycerol provided performance suitable for 3 to 6 min separations at (60 V/cm) in fsPAG-EMSA current stability, separation performance, and reliability. Unit-to-Unit Variation Across a Large fsPAGE Array. To realize a high-throughput EMSA platform, we fabricated a 96-plex 20% T 3.3% C fsPAG array measuring 60 mm × 70 mm (Figure 5). The array is comprised of 8 parallel fsPAG lanes, each housing 12 separation units in series with a 4.5 mm unitto-unit distance. To electrically address the array, graphite electrodes were placed atop electrode wicks orthogonal to each fsPAG lane and ∼2 mm away from the first and last separation unit in each lane. In previous work, we observed significant unit-to-unit variation in analyte migration distance on large fsPAG arrays, especially in units on the periphery of the device.27 To understand and address migration variation, we measured the migration distance of a scalar (fluorescently labeled 500 nM OVA*) at a set time and tested significance of unit-to-unit variation with an ANOVA test (Figure 5A and see the Supporting Information for details). In particular, we
Figure 5. Unit-to-unit variation of migration distance on the 96-plex fsPAGE. (A) Fluorescence imaging of 96 concurrent OVA fsPAGE. A gel defect was detected in Row 5, Col 6, resulting in a “band bowing” on this unit (excluded from calculations). 96 units were grouped into regions. Central region group, a; other peripheral groups, b−f. F value for migration distance: a−b, 438; a−c, 121; a−d, 205; a−e, 0.14; a−f, 0.7. Significant variations were observed for a−b; a−c; and a−d (squared in red). No significant variations were observed for other groups (squared in green), α = 0.05. (B) Optimized fsPAG design with offset between electrodes and fsPAG units. Central region group, a; peripheral groups, b−d. F values: a−b, 1.67; a−c, 2.21; a−d, 0.05; no significant variations were observed (squared in green), α = 0.05. Fcritical = 4.00 for ANOVA on 96-plex fsPAG.
sought to assess the spatial variation in migration distance between peripheral groups and the central region (groups F
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assigned in Figure 5A). For each peripheral group, ANOVA was performed to analyze its variation against the central region group a, where no spatial variation is present. In the present ANOVA study with 96-plex fsPAGE, the Fcritical value is 4.00 (α = 0.05), meaning any calculated F value greater than 4.00 indicates significant difference in migration distance. For fsPAG lanes at the top and bottom of the array, no significant variation in migration distances were observed as compared to migration distances observed in the central array region (F values 0.14 and 0.70, respectively). In contrast, regions adjacent to the electrodes showed significant variation in scalar migration distance. As compared to the central region of the array, the first and second columns adjacent to the cathode electrode supported a higher scalar velocity (F values 438 and 121, respectively) and the 12th column directly adjacent to anode supported a lower scalar velocity (F value 205). We hypothesized that electrolysis during electrophoresis altered the pH proximal to each electrode. At the cathode, electrolysis should generate excess OH− ions and elevate the local pH causing proteins to increases in negative charge and mobility. Conversely the anode would see generation of excess H+ ions and lower the local pH, thus reducing protein charge and mobility. While there were several possible approaches to address pH polarization, including increasing buffering capacity or implementing buffer reservoirs, we opted to simply redesign the electrode region of the 96-plex fsPAG array to offset the graphite electrodes from the first and last separation columns. The modified electrode regions increased the array area from 4200 mm2 to 7000 mm2. With the offset electrode placement (Figure 5B), ANOVA reported no significant region to region variation in migration distance for the scalar (F = 1.67, 2.21, and 0.05 for columns 1, 2, and 12, respectively). fsPAG-EMSAs for c-di-GMP Riboswitch on the 96-Plex fsPAG. Having established the optimized fsPAG conditions, the 96-plex fsPAG-EMSAs were applied in high-throughput detection of binding of the Vc2 aptamer to c-di-GMP. On the basis of previous in-chip EMSAs as well as previously characterized Vc2 binding rates,36 the expected interconversion rate between bound and unbound RNA is slower than the rate of electromigration, indicating that the EMSAs will resolve the binding-induced conformation change into two distinct bands. The separate bands visualized in the slow interconversion regime allow for the Kd or a standard curve of the system to be calculated by the relative intensity of each peak.37 To analyze ligand binding to the Vc2 aptamer, we constructed a 96-plex 20% T 3.3% C fsPAG for highthroughput riboswitch fsPAG-EMSAs (Figure 6A). The array has 9 mm horizontal well-to-well spacing and 9 mm lane-tolane distance such that a standard multichannel pipet provides a suitable macro-to-micro interface. The binding reactions contained 1 μM RNA and 0−900 nM c-di-GMP using 500 nM of TI* as an internal standard. On the basis of the high affinity of the Vc2 aptamer for c-di-GMP (Kd ∼ 11 pM),36 the high RNA concentration used here results in the generation of a standard curve rather than a Kd curve, which would require the concentration of one reagent to be below the Kd. Electrophoresis was performed for 2.5 min followed by fluorescence scanning of the array. The observed mobility shifts demonstrate the successful application of 96 parallel EMSAs in 3 min. For assays that require substantially longer separation times (>5 min), we are investigating a semienclosed fsPAG structure, which retains the facile open format while potentially providing longer separation duration. Additionally, the cumbersome manual loading process significantly limits the overall throughput of the assay (∼5 min loading out of 10 min total assay). Automation of fsPAGE loading with robotic sample loading systems (
